Micro lysis-analysis process to measure cell characteristics and diagnose diseases

ABSTRACT

A microphotolysis and microphotoanalysis process applying precise quantities of focused light energy to a precise microscopic area of a very small cell sample to activate a chemical agent which permits measurement of cell fragility.

This application is a Continuation-In-Part of U.S. patent applicationSer. No. 07/969,764 filed on Oct. 30, 1992.

BACKGROUND OF THE INVENTION

Under various cell environments, most biological cells can drasticallychange their shape (called cell "deformability") without structuraldamage to the cell membrane, without a loss in cell contents to the cellenvironment, and without a change in the chemical function of the cellmembrane. Under other cell environments, the cell membrane can losechemical function, can suffer structural damage, and can rupture to losecell contents to the cell environment (called cell fragility). Thus,cell deformability and cell fragility are two independentcharacteristics of a cell membrane. Cells can have any combination of ahigh-to-low deformability and a high-to-low fragility. However, mostnormal cells have high deformability and moderate-to-low fragility.

Deformability is one important characteristic for some cells such as"sensory receptors" which are normally stationary in the cellenvironment and for other cells such as blood and lymph cells whichnormally move in the cell environment. Cell fragility is an importantcharacteristic for almost all normally stationary and normally movingcells because changes in the cell environment can cause considerableamounts of water to move into cells to rupture more fragile cells.Changes in cell fragility can change the ability of a cell to performits normal function in a normal or abnormal cell environment. Forexample, an abnormally high fragility of red blood cells will lead topremature rupture of many red blood cells in a normal blood environmentwhich will reduce the circulating pool of red blood cells and willreduce the red blood cell transport of oxygen to tissues. Yet, thesehighly fragile cells can have normal cell deformability. Thus, fragilitymight clinically be a more important cell characteristic thandeformability.

Cell fragility is altered by many conditions such as cell age, durationof blood-bank storage, treatment with a variety of membrane-bindingdrugs, and progression of membrane or hemoglobin-related diseases suchas diabetes and sickle-cell anemia, respectively. Thus, a rapid, highlyaccurate, and easily applied method is needed for clinical measurementsto assess cell fragility as an index of "cell strength" which is definedas the degree to which the cell membrane can maintain its structuralintegrity and its chemical function in a normal and an altered cellenvironment. Most current methods for assessing cell strength primarilycreate mechanical forces on cells to assess either the deformability orthe fragility of these cells. These methods include Osmotic-GradientEktacytometry, and Cell Filtration, Micropipette Suction, and OsmoticFragility tests.

Osmotic-Gradient Ektacytometry is one method that applies mechanicalforces to measure a combination of cell deformability and cellfragility. This method uses a viscometer device to measure shape changeswhich are induced in red blood cells by various speeds of rotation(called applied shear stress) of the test cells with different osmoticsolutions in the cell environment. The osmotic-spectrum curves which areproduced by Ektacytometry are highly variable for the same test-cellsample due to small changes in sample ambient temperature, pH, andplasma osmolality. Thus, these osmotic-spectrum curves are complex andvery difficult to use for interpretation of changes in celldeformability. As a result, Ektacytometry currently requires verysophisticated equipment, extensive operator training, and highlycontrolled test conditions to obtain one measurement value for thecombination of cell deformability and fragility. This makesEktacytometry usable only in a few research laboratories and not in aclinical setting.

Other methods such as Cell Filtration and Micropipette Suction alsorequire the external application of a mechanical force to cells butthese methods primarily measure cell deformability by forcing(filtering) cells through various size pores or by mechanical aspirationof these cells into micropipettes of fixed tip size and taper. Thesemethods also require very sophisticated equipment and substantialoperator training; and they are extremely time consuming to obtain ananalysis of only a relatively few cells in each test-cell sample. Thus,these methods have also not been accepted into general clinical use.

Assessment of cell deformability in the Cell Filtration method has beenimproved by measurements of electrical impedance (Hanss et al, U.S. Pat.No. 4,835,457) and measurements of time (David D. Paterson, U.S. Pat.No. 4,491,012) during the application of external mechanical force(pressure) to force red blood cells to pass through either an artificialmembrane filter or a foil system (Helmut Jahn, U.S. Pat. No. 4,797,606).These measurement methods are extremely sensitive to manufacturingtolerances on the filter or foil and both of these measurement methodsprimarily assess only cell deformability and not cell fragility. Thus,these measurement additions to the Cell Filtration method have also notfound their way into common clinical use.

The Osmotic Fragility method was one of the earliest techniques that wasdeveloped for assessment primarily of red blood cell fragility ratherthan deformability, and it is one of the few methods currently inclinical use. The Osmotic Fragility method is time-consuming, and itrequires multiple blood handling steps, and relatively large volumes ofblood samples. The Osmotic Fragility method uses exposure of bloodsamples to a large range of salt concentrations to produce a large rangein the osmotic pressure for the cell environment. The osmotic pressuremechanically forces water into cells to swell cells to the point of cellmembrane rupture.

The osmotic pressure method has been adapted by Groves and Rodriguez(U.S. Pat. No. 4,535,284) to apply high frequency and low frequencyelectrical currents to detect the percent of red blood cells that arealtered during application of osmotic mechanical forces over a largerange of osmotic cell environments. This use of high frequency and lowfrequency electrical currents is combined with a Coulter Counter® toclassify individual red blood cells as normal or abnormal.

Overall, the osmotic mechanical gradient method and various refinementsto this method can only detect relatively large changes in cell membranefragility because this osmotic-based mechanical method provides noinformation about the rate of cell lysis (which occurs when the cellmembrane ruptures). The lack of method sensitivity to mild or moderatechanges in cell fragility has led to the clinical use of the osmoticmechanical method only for diagnosis of one disease called hereditaryspherocytosis.

In contrast to methods that apply external mechanical forces mostly tomeasure cell deformability or in one case (osmotic gradients) to measurecell fragility, there is a chemical method which changes the mechanicalcharacteristics of cell membranes to assess cell membrane fragility. Itwas first shown some fifty years ago that some chemicals can be changedby light (called photoactivation) to induce the rupture or breakup ofred blood cell membranes (called hemolysis) in a test tube. Since then,this basic process (called photohemolysis) has been extensively studiedin a variety of test-tube experiments.

The mechanism for photohemolysis is oxygen dependent and is thought toinvolve the generation of singlet oxygen with the subsequent oxidationof proteins in the red blood cell membrane. It has been suggested thatthis protein oxidation leads to the creation of extra water channels inthe cell membrane. This would increase passive cationic exchange acrossthe cell membrane to give a subsequent influx of water into the cell toswell the cell to the point of hemolysis. It has been suggested thatphotohemolysis could also involve peroxidation of the lipid layers inthe cell membrane. This peroxidation appears to limit the ability ofmolecules to move (called membrane fluidity) in the lipid bilayer of thecell membrane, which then appears to limit the ability of the cell toundergo shape changes (cell deformability), since shape changes innormal cells appear to depend on membrane fluidity in the lipid bilayer.

Thus, there is considerable scientific evidence to show that certainchemical agents can be photoactivated to create cell-attack agents whichdisrupt red blood cells by altering either the protein or the lipidlayer of the cell membrane. These alterations in cell membrane structurepermit water inflow to increase internal cell volume until the "internalcell pressure" ruptures the cell membrane (called cell fragility)sufficiently to cause the loss of cell contents (called lysis in generalor hemolysis in the case of red blood cells).

Currently, a limited number of chemical agents have been identified aspotential cell-attack agents. Previous research has not yet discoveredan acceptable method for use of these cell-attack agents to reproduciblycreate photohemolysis. Previous uses of these cell-attack agents havebeen limited by requirements for long photoactivation periods, longanalysis times, test-cell isolation from the original blood sample,multiple blood samples and high volume (milliliter) samples, and by atest result that gives only a single measurement parameter for eachsample.

The long photoactivation periods have been a major limitation. Forexample, photoactivation of red blood cells incubated with 0.1 mM ofprotoporphyrin as the cell-attack agent requires approximately twentyminutes of photoactivation exposure time to achieve only a modest 20%hemolysis in the cell sample; a 100% hemolysis requires a 25-minute orlonger photoactivation exposure period with this agent. Similarly, morethan twenty minutes of photoactivation exposure is needed withpheophorbide as the cell-attack agent to give approximately 90%hemolysis in the cell sample. Photoactivation periods of 3-4 hours arerequired with eosin-isothiocyanate as the cell-attack agent to givemaximal hemolysis which only then occurs approximately 11 hours afterthe photoactivation period. These long photoactivation periods producehighly variable hemolysis rates and hemolysis levels which have limitedthese photohemolysis techniques to research use.

A few researchers have previously attempted the use of a lightscattering device to detect the presence of significant photohemolysis.However, this device is very sensitive to very small changes in redblood cell concentrations, and this device requires a monolayer of redblood cells with a red cell concentration of less than 0.00025%. Suchconcentrations have not been practicable to achieve even by currentmodern micropipetting systems. In addition, the use of a cell monolayerand the light scattering measurement have also been limited by a longphotoactivation and hemolysis measurement period since 100% hemolysis ofa cell sample requires four hours with phloxine B as the photoactivatedcell-attack agent.

Current clinical methods rely on osmotic mechanical forces over a wideosmotic range to create hemolysis, and these methods require milliliter(i.e. macro) quantities of blood volumes to determine an osmolalityrange (equivalent to internal water pressure) within which hemolysisoccurs. These Osmotic Fragility tests are generally performed on samplevolumes in test tubes or cuvettes, and parallel light is required fordetection of the presence or absence of significant hemolysis. Thesecurrent clinical hemolysis methods cannot separately measure changes incell deformability and cell fragility, cannot distinguish changes incell membrane fragility due to alterations in the protein layer of themembrane from fragility changes due to alterations in the lipid layersof the membrane, and cannot determine "a rate of hemolysis" which wouldprovide a more sensitive hemolysis test for clinical use.

Both the current Osmotic Fragility clinical method and thePhotohemolysis research method require multiple cell samples, multiplesample dilutions, sample centrifugation, and separate sample analysis ina spectrophotometer to give a single measurement of a parameter calledPercent Cell Hemolysis at one point in time as an assessment of cellmembrane fragility. Thus, these current clinical and research methods donot provide simultaneous energy-dose dependent and time-dependentmeasurements of photoactivation and hemolysis relationships from one,small volume (i.e. micro), blood sample.

SUMMARY OF THE INVENTION

The present invention includes a new "micro-lysis" process which appliesprecise measurable quantities of focused energy to a precise microscopicarea of a very small cell sample (a cell micro-sample of less than 25 μl) to activate a chemical agent (called the energy-activated cell-attackagent) which then attacks the cell membranes only in the microscopicarea of the cell micro-sample. The present invention also includes a new"micro-analysis" process which measures, as a function of time, theprecise degree of cell membrane rupture which is caused by theenergy-activated cell-attack agent in the microscopic area ofcell-attack. The combination of these two processes give a quantitative,time-related, energy-dose dependent, multivalue measure of the cellmembrane property that is commonly called cell fragility.

The present invention (called the Micro Lysis-Analysis Process) hasseveral advantages over other current methods which give relativelyimprecise single-value estimates of cell fragility. The MicroLysis-Analysis process is unique in its use of microscopic focusedenergy to activate the cell-attack agent only in a very small definedmicroscopic area of a cell micro-sample. Thus, independent cell-attackin multiple separated microscopic areas can be initiated to obtainmultiple cell fragility measurements in a single micro-sample, withother microscopic areas left as baseline unactivated cell-attack samplereferences. Each microscopic area of a micro-sample can also be analyzedbefore energy activation of the cell-attack agent in each respectivemicroscopic area to give a local microscopic baseline reference forsubsequent determination of the lysis response in each respectivemicroscopic area. In contrast to other current methods, these advantagesof the present invention provide multiple "energy-dose lysis-response"relationships to give very precise time-related measurements of cellmembrane fragility in a single micro-sample.

Unlike other current methods, the novel use in the present invention offocused energy on a small microscopic area of a cell micro-sample onlyrequires relatively short energy-exposure times of 1 to 10 minutes toactivate the cell-attack agent, and a relatively short time, dependingon the cell population, of 30 to 60 minutes to obtain complete celllysis. Thus, the present invention provides a unique and completemicro-lysis response curve to measure the rate as well as the finaldegree of cell lysis within a total measurement time of one hour or lesson a single micro-sample of 25 microliters (μl) in volume.

In the present invention, precise quantities (doses) of various testchemicals can be added to the cell micro-sample to provide adose-response test for the effect (efficacy) of those chemicals to altercell fragility. Thus, the present invention can provide a unique andcomplete dose-response analysis for six doses of a test chemical on atotal blood sample of 150 μl in volume.

Unlike other current methods, the present invention is a new processwhich does not require cell isolation from the original blood sample,multiple blood samples, or further cell manipulation after themicro-sample is placed in the sample-carrier. The present invention iseasy to perform with easy-to-conduct tests of reproducibility byseparate energy-activation in multiple microscopic areas of eachmicro-sample. The present invention is a new process which gives amicro-lysis that depends only on the level of energy-activation and onthe concentration and formulation of the cell-attack agent. The presentinvention is a new process that is not affected (as are other methods)by variations in sample temperature which can increase Brownian cellmotion to confound other lysis measurements, and is not affected bynon-specific effects of other blood constituents such as plasma in thecell micro-sample. Thus, the present invention is a new process whichcreates a very specific energy-level related cell lysis only in the areaof focused energy-activation of the cell-attack agent.

The present invention is a new process that can measure alterations incell membrane fragility due to changes in membrane characteristics thatare created by a cell-attack agent that remains and becomes activatedonly outside the cell (an extracellular cell-attack agent). The presentinvention is a new process that can also measure alterations in cellmembrane fragility due to changes in internal cell structures that arecreated by a cell-attack agent that crosses the cell membrane to attachto internal cell structures where the cell-attack agent becomesenergy-activated. Thus, the present invention is a new process which canprovide first-ever detection of systemic diseases that alter only thecell membrane, systemic diseases which alter only the internal cellcomposition, and detection of alterations in cell fragility due totreatments of cells with test chemicals.

The energy level for activation of an intracellular cell-attack agentwill in part depend on the anti-oxidant status of the cell. Many cellssuch as red blood cells usually carry oxygen and these cells maintain awell developed anti-oxidant system to protect the cell againstauto-oxidation. Other current methods to assess cell fragility cannotalso assess anti-oxidant activity because these other methods do notinduce oxidation reactions within the cell. The present invention is anew process which can measure the difference in cell fragility due toenergy-activation of separate external and internal cell-attack agentsto determine the status of the anti-oxidant system in the cell.

The present invention is a new process (called a Micro Lysis-AnalysisProcess) which uses a generally known configuration of generallyavailable equipment, new calibration procedures, new solutions, new cellpreparations, and a new micro lysis-analysis procedure in a critical newsequence of steps to measure cell fragility in microscopic regions of acell-containing micro-sample.

Accordingly, it is an object of the present invention to utilize agenerally known configuration of microscope, light source, lightcontrol, television imaging, and video recording equipment to providemicroscopic images of cell samples.

It is a further object of the present invention to provide a newmicroscope calibration procedure to provide exposures of cells toquantitated, focused, frequency-specific energies.

It is a further object of the present invention to utilize a generallyknown selection procedure to obtain a cell specimen.

It is a further object of the present invention to utilize preparationof a standard buffer solution to provide a standardized environment forthe cell specimen.

It is another object of the present invention to provide a new methodfor a quantifiable cell-attack agent in different microscopic areas ofmicro-samples of the cell specimen.

It is yet another object of the present invention to provide a newmethod for preparation of the cell specimen to give a standardizedmixture of cells and a cell-attack agent in a standardized solution(buffer) environment.

It is a further object of the present invention to provide a newmicro-sample preparation for subsequent measurements of cell rupture.

It is a further object of the present invention to provide a newmicro-lysis procedure for precision energy activated cell-attack torupture the cells in the micro-sample in a new standardized manner.

It is yet a further object of the present invention to provide a newprocedure for micro-analysis of micro-sample images to measure thetime-sequenced rupture of cells during energy-activated cell-attack inmicroscopic regions of the micro-sample.

These and other improvements will be better understood on reading thedescription which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be had uponreference to the following description in conjunction with theaccompanying drawings, wherein:

FIG. 1 is a plot showing curves for the percentage number of survivingintact human red blood cells (measured as a relative Response-AreaOptical Density) in two rectangular microscopic areas of a cellmicro-sample as a function of time from the beginning of a second-energyexposure of 245 Joules/cm² (open circles) which activates thecell-attack agent to a low degree in one rectangular microscopic area tocreate a moderate rate of micro-lysis over a 40-50 minute period in thatarea of the micro-sample and a second-energy exposure of 401 Joules/cm²(filled circles) which activates the cell-attack agent to a higherdegree in a second rectangular microscopic area to create a higher rateof micro-lysis over a 25-30 minute period in that second area of themicro-sample.

FIG. 2 is a plot showing a repeat of the micro-lysis curve (opencircles) of FIG. 1 for second-energy cell-attack activation at 245Joules/cm² in one rectangular microscopic area of the cell micro-sampleand gives a Response-Area Optical Density curve as a function of timefrom the beginning of a second-energy cell-attack activation of 638Joules/cm² (filled circles) to create micro-lysis in a third rectangularmicroscopic area of the micro-sample that gave FIG. 1.

FIG. 3 is a plot showing a repeat of the curve (open circles) of FIG. 1as the graphical basis for definition of:

1) the Maximal Micro-lysis Response (MR) as the zero-time Response-AreaOptical Density (ZROD) minus the Minimum Plateau Response-Area OpticalDensity (PROD);

2) the Percent Maximal Response (MR%) as 100 times MR divided by ZROD;and

3) the % Response at a specific time t (% Response (t)) as 100 times thequantity, ZROD minus the Response-Area Optical Density at time t(ROD(t)), that resultant quantity divided by MR.

FIG. 4 is a plot showing the three micro-lysis % Response curves whichare generated by application of the definitions in FIG. 3 to the threeResponse-Area Optical Density curves in FIG. 1 and FIG. 2.

FIG. 5 is a plot showing a repeat of the micro-lysis % Response curve(open circles) of FIG. 4 as the graphical basis for definition of:

1) the response half-time (T_(1/2)) as the period from the beginning ofcell-attack activation to the time of the 50-percent response value, and

2) the largest slope of the % Response Curve as the Slope at timet=T_(1/2).

FIG. 6 is a plot showing the % Response curve (mean±SEM) for micro-lysison day 1 at 7 hours (circles), day 2 (squares), day 3 (triangles), day 4(diamonds), and day 5 (inverted triangles) after collection of red bloodcells from four rabbits.

FIG. 7 is a plot showing the % Response curves (mean±SEM) for the effectof cell-attack activation of 105 Joules/cm² (open squares), 126Joules/cm² (filled squares), 159 Joules/cm² (open triangles), 168 J/cm²(filled triangles), 210 J/cm² (open diamonds), and 210 J/cm² (filleddiamonds) on rabbit (N=3) red blood cells.

FIG. 8 is a plot showing the % Response Curves (mean±SEM) formicro-lysis of 3% (circles), 4% (triangles), and 5% (squares)concentrations of rabbit (N=4 ) red blood cells.

FIG. 9 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=3) red blood cells that had been separated fromwhole blood (circles) and for red blood cells in whole blood thatcontained plasma and other cells such as platelets and white blood cells(triangles).

FIG. 10 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=4) red blood cells on day 5 (circles), on day 5after the red blood cells had been washed and reconstituted with bufferand the cell-attack agent FITC-dextran (diamonds), and on day 5 afterthe red blood cells had been washed and reconstituted with buffer butnot the cell-attack agent FITC-dextran (squares).

FIG. 11 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=4) red blood cells that had been prepared instandard buffer with glucose and albumin (squares), buffer with glucosebut not albumin (filled diamonds), buffer with albumin but not glucose(triangles), and buffer without albumin and glucose (circles).

FIG. 12 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=6) red blood cells which had been previouslyincubated for one hour with a test chemical diamide at a concentrationof 5.0 milliMolar (diamonds), 0.5 mM (squares), 0.05 mM (circles), or0.00 mM (filled triangles), with cell-attack activations of 180 J/cm²(right panel) and 90 J/cm² (left panel).

FIG. 13 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=5) red blood cells which had been previouslyincubated for 20 minutes with the anesthetic Chlorpromazine at aconcentration of 100 microMolar (circles), 30 μM (diamonds), 10 μM(squares), and 0 μM (filled triangles), with cell-attack activations of180 J/cm² (right panel) and 90 J/cm² (left panel).

FIG. 14 is a plot showing the % Response curves (mean±SEM) formicro-lysis of rabbit (N=6) red blood cells which had been previouslyincubated for one hour with the test chemical glutaraldehyde at aconcentration of 0.02% (circles with dashed line), 0.01%, (diamonds),0.005% (triangles), 0.0025% (squares with dashed line), 0.00125%(circles), and 0.000% (filled triangles).

FIG. 15 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves for micro-lysis ofred blood cells from 5 normal control Sprague-Dawley laboratory rats (C)at the cell-attack activation of 248±3.6 J/cm², 3 streptozoticin-inducedinsulin-dependent diabetic Sprague-Dawley rats (D) at 245±4.1 J/cm², and6 diet-induced hypercholesterolemic Sprague-Dawley rats (H) at 249±2.7J/cm².

FIG. 16 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves for micro-lysis of0.5% to 3.0% concentrations of human (N=3) red blood cells. One subjectreceived a Cell-attack activation of 549±0.3 J/cm², the second subjectreceived 340±1.1 J/cm², and the third subject received 198±1.3 J/cm² forall five cell concentrations.

FIG. 17 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves for micro-lysis ofhuman (N=5) red blood cells which had been previously incubated at 4° C.FOR ONE HOUR with the therapeutic drug pentoxifylline (PTFX) atconcentrations of 10 milliMolar (filled bars), 1 mM (striped bars), and0 mM (clear bars). One subject received a cell-attack activation of658±6.6 J/cm², three subjects received 329±1.9 J/cm², and one subjectreceived 196±0.7 J/cm² for all three pentoxifylline concentrations.

FIG. 18 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves for micro-lysis ofhuman (N=5) red blood cells which had been previously incubated at 4° C.FOR TWENTY-FOUR HOURS with the therapeutic drug pentoxifylline (PTFX) atconcentrations of 10 milliMolar (filled bars), 1 mM (striped bars), and0 mM (clear bars). One subject received a cell-attack activation of641±3.3 J/cm², three subjects received 330±1.7 J/cm², and one subjectreceived 205±0.7 J/cm² for all three pentoxifylline concentrations.

FIG. 19 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves at cell-attackactivations of A=243±2.9 J/cm² (clear bars), B=400±3.6 J/cm² (stripedbars), and C=642±4.0 J/cm² (filled bars) for micro-lysis of red bloodcells from four female and three male African-Americans who had noclinical indicators of sickle-cell disease.

FIG. 20 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves at cell-attackactivations of A=247±1.1 J/cm² (clear bars), B=395±5.5 J/cm² (stripedbars), and C=643±4.0 J/cm² (filled bars) for micro-lysis of red bloodcells from 2 female and 3 male African-Americans with clinical diagnosisof sickle-cell disease but not hemoglobin-F abnormality.

FIG. 21 is a bar graph showing the Percent Maximal Response (MR%) andthe T_(1/2) and Slope values of the % Response curves at cell-attackactivations of A=253±2.8 J/cm² (clear bars), B=399±4.6 J/cm² (stripedbars), and C=661±2.1 J/cm² (filled bars) for micro-lysis of red bloodcells from 1 female and 1 male African-American with clinical diagnosisof sickle-cell disease and hemoglobin-F abnormality.

FIG. 22 is a photomicrograph showing the rectangular microscopic area ofcell-attack for a cell micro-sample before cell-attack activation asseen in the top left panel, 20 minutes after the start of cell-attackactivation as seen in the top right panel, 40 minutes after the start ofcell-attack activation as seen in the bottom left panel, and 60 minutesafter the start of cell-attack activation as seen in the bottom rightpanel.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Process Requirements

The present invention is a process that requires, as a minimum, amicro-sample which contains, as a minimum, the cells of interest and achemical agent that can be energy-activated to become a cell-attackagent; a device to hold the microsample; a device to focus energyprecisely on selected microscopic areas of the micro-sample; a device toproduce a first-energy form which is used for the analysis of cellrupture in the selected microscopic areas of the micro-sample; a deviceto produce a second-energy form which is used to energy-activate thecell-attack agent in the micro-sample; a method to ensure the deliveryof a precise amount of the first-energy form to the micro-sample; amethod to ensure the delivery of a precise amount of the second-energyform to the micro-sample; a device to measure the amount of thefirst-energy form that is transmitted through or is reflected from or isscattered from the micro-sample as a function of time; and a method toquantitate the effect of the time-dependent transmitted, reflected, orscattered first-energy measurement of cell rupture in the micro-samplein relationship to the time-dependent amount of the second-energy formthat is delivered to the micro-sample.

A number of currently known chemicals are capable of energy activationto become cell-attack agents. It is contemplated that many more suchchemicals will be identified in the future. Each known chemical requiresenergy activation in a specific energy band (i.e. a specific frequencyand quantity of energy) to become an effective cell-attack agent. It iscontemplated that future such chemicals will be identified to havespecific energy bands across a broad range of the overall energyspectrum. The second energy form to be used in the present invention isdependent on the chemical that is selected to be the cell-attack agentin each application of the present invention.

Likewise, each currently known type of cell has a specific energy band(i.e. a specific frequency) in which each respective cell type absorbs,reflects, or fluoresces that energy. It is contemplated that future celltypes will be identified to have specific energy bands across a broadrange of the overall energy spectrum. The first-energy form to be usedin the present invention is dependent on the cell type that is selectedto be analyzed for cell fragility in each application of the presentinvention.

The following elements of the preferred embodiments for the presentinvention are described for, but are not limited to, a selection offluorescein isothiocyanate (FITC) as an example of the cell-attack agentand for selection of the red blood cell (erythrocyte) as an example ofthe cell type. FITC has an absorption energy band centered atapproximately 480 nanometers, and the red blood cell has an absorptionenergy band above 500 nanometers. Both of these absorption bands are inthe visible (i.e. light) energy range of the overall energy spectrum.Thus, as a result of those specific selections as examples, thefollowing describes, but is not limited to, a transillumination pathwayof a light microscope for the focus of the first-energy form whichcontains significant energy at energy frequencies above 500 nanometersto be absorbed by the red blood cell as a selected cell type; anepi-illumination pathway of the same microscope for the focus of thesecond-energy form which contains significant energy at the energyfrequency of 480 nanometers to activate FITC as a selected cell-attackagent; and the appropriate energy-measuring equipment for the process tomeasure energy levels (i.e. quantities) in the visible (i.e. light)energy band at the plane of the micro-sample.

Equipment Configuration

Although any microscope having a 75-300 W arc lamp can be used in thepresent invention, a Zeiss model 20-T light microscope having a 100 Wmercury arc lamp is utilized in the preferred embodiment to provide thesecond-energy form by epi-illumination of the cell micro-sample throughthe microscope objective lens. A halogen lamp in the range of 50-300 W,preferably a 100 W halogen lamp, is used to provide the first-energyform by transillumination of the cell micro-sample through themicroscope substage condenser. A television camera with external controlof target voltage (either a charged-coupled-device (CCD) or asilicon-intensified-target (SIT) camera) is equipped with a microscopeeye-piece attachment for adequate microscope-image capture,videorecording, video monitor display, and analysis of micro-lysis. Astandard videocassette recorder such as a Panasonic model 6200, atime-date video-insertion generator such as a Panasonic model 810, and amoderate resolution television monitor (at least 600 horizontal linespreferred) are used to record the images of a selected microscopic areaof the cell-containing micro-sample for analysis of the first-energyimages on-line or off-line at a convenient time.

The preferred embodiment of the present invention uses alight-microscopy objective lens (UMK 50X Leitz) with a 0.60 numericalaperture having a variable built-in lens diaphragm, a microscope tubefactor of 1.25 X, an X-Y coordinate movable microscope stage such asthat found on a Zeiss 20-T standard microscope, and a brightfieldsubstage condenser such as that found on a Zeiss 20-T standardmicroscope, all of which are readily available commercial components. Amicroscope filter cube (Leitz model I-2 preferred) is utilized with aband-pass filter having a preferred range of 450-490 nm, a dichroicmirror preferably with a transmission above 510 nm, and a long-passbarrier filter preferably with a transmission above 515 nm which arechosen to, correspond to the absorption and emission frequencies of thechosen energy-activated cell-attack agent such as FITC.

The second-energy form is provided by an epi-illumination pathwaythrough the microscope. This epi-illumination pathway has provisions forthe positioning of a variable circular diaphragm, a fixed rectangulardiaphragm (4.0×3.5 millimeter aperture preferred), and a neutral densityfilter carrier between the epi-illumination second-energy source and themicroscope filter cube to permit graded and known decreases in theamount of activation energy which impinges as a second-energy form atthe cell micro-sample on the microscope stage.

The first-energy form is provided by a transillumination pathway throughthe cell micro-sample. This transillumination pathway has provisions forthe positioning of a circular field diaphragm (22 mm diameter aperturepreferred), a circular field energy-limiter (7.5 mm diameter aperturepreferred), and a color filter (green preferred for red blood cells)between the transillumination first-energy source and the substagemicroscope condenser to permit and control the amount of thefirst-energy form which impinges at the cell micro-sample on themicroscope stage.

Microscope Calibration Procedure

The present invention includes a new microscope energy-calibrationprocedure to provide micro-sample exposures to specific amounts offrequency-specific energies which are quantitated as "energy power"multiplied by the "time of energy exposure." In the following procedure,the critical steps are the independent adjustments of theepi-illumination and transillumination energy pathways to give aspecific energy power from each pathway to a specific area at the cellmicro-sample when the cell micro-sample is "in focus" on the microscopestage. Energy power at any specific point in an energy path can bemeasured by any of several instruments (such as an EGG model 580radiometer). The preferred instrument for energy in the visible energyband is a model 61 United Technology (UT) Optometer which measuresenergy power in the electrical power equivalents of milliwatts.

To begin calibration of the transillumination pathway, a glassmicroscope slide is covered with a glass coverslip and is positioned onthe stage of the microscope. The epi-illumination pathway is blocked byclosure of the variable circular diaphragm. After adjustment of theobjective lens to bring the glass slide into focus on themicroscope-attached television camera tube, the field diaphragm of thetransillumination pathway is adjusted to a nearly closed position (apin-hole opening) and the substage condenser is raised to bring thepin-hole opening of this field diaphragm into focus. The x-y adjustmentsof the substage condenser are then used to center the pin-hole openingof this field diaphragm in the microscope field-of-view.

The power supply for the transillumination first-energy pathway isadjusted to approximately 9.2 volts. The energy power for this pathwayis measured at the exit of the field energy-limiter, and the fielddiaphragm is opened until the energy power at the exit of theenergy-limiter plateaus to give a value of 1.08 to 1.88 (1.48 preferred)milliwatts. If the plateau energy power is too high or too low at theenergy-limiter, the voltage of the first-energy power supply should beadjusted. The energy power is then measured at the glass slide on themicroscope stage, and the built-in diaphragm of the substage condenseris partially closed to give 0.044 to 0.052 (0.048 preferred) milliwattsof transilluminated energy power at the glass slide.

To begin calibration of the epi-illumination second-energy pathway, adrop of fluorescein diacetate (1.0 mg/ml concentration) is placed on aglass microscope slide and is covered with a glass coverslip. The slideis placed in a sample carrier which is positioned on the stage of themicroscope. The transillumination pathway is blocked by placement of anopaque filter on the field limiter. The variable circular diaphragm ofthe epi-illumination pathway is slightly opened to focus a fluorescentimage of a pin-hole in this diaphragm on the microscope-attachedtelevision camera tube. The diaphragm-holder adjustments are used tocenter the fluorescent image of this circular diaphragm in thetelevision field-of-view.

The circular diaphragm is then opened fully and the fixed rectangulardiaphragm is inserted into its carrier in the epi-illumination pathway.The holder adjustments for the rectangular diaphragm are adjusted tocenter the fluorescent image of this rectangular diaphragm in thetelevision field-of-view. The second-energy source adjustments in thesource housing for the epi-illumination second-energy pathway are thenused to produce an even fluorescence intensity across the televisionimage of the rectangular diaphragm. The circular diaphragm of theepi-illumination pathway is closed completely and then opened to thepoint where the rectangular diaphragm is imaged as a uniform fluorescentrectangular area without any reflections of stray energy in thetelevision field-of-view.

The energy power of the epi-illumination second-energy pathway ismeasured at the focal plane of the objective lens (which corresponds tothe position of the coverslip in the sample carrier on the microscopestage when that coverslip is in focus in the television field-of-view),and this energy power is adjusted to a value of 0.39 to 0.41 (0.40preferred) milliwatts by partial closure of the diaphragm in theobjective lens. (If the energy power is too low at this point, theepi-illumination second-energy source has aged and should be replaced.)

Neutral Density (ND) filters are then placed one at a time in theepi-illumination pathway to provide calibration of graduated decreasesin the epi-illumination second-energy power at the focal plane of theobjective lens. Nominal epi-illumination second-energy power values are0.30-0.32 milliwatts for an ND of 0.1, 0.18-0.20 milliwatts for an ND of0.3, and 0.10-0.12 milliwatts for an ND of 0.6.

The amount of epi-illumination, which is the second-energy form used toenergy-activate a cell-attack agent to rupture cells in a micro-sample(described below) on the microscope stage, is quantitated as an energydensity (ED) which equals epi-illumination energy power at themicro-sample, multiplied by time of epi-illumination energy exposure,divided by the micro-sample area of epi-illumination energy exposure.For the above-described equipment configuration and calibrationprocedure, the area of epi-illumination energy exposure is determined bythe fixed rectangular diaphragm which has a projected image area of0.000175 cm² (140.91 μm×124.24 μm) at the focal point of the microscopeobjective lens.

A common unit for energy density is Joules/cm² where one Joule equalsthe total energy that is released in one second by a one-ampere currentflowing through a one-ohm resistor (which is electrically equivalent toone watt or 1000 milliwatts of power for one second). For theabove-described calibration procedure, the epi-illumination energy poweris measured as milliwatts (mw) by the UT Optometer, and epi-illuminationexposure time is measured in 0.01 minute increments by the time-datevideo-insertion generator. The following formula is used to convertthese measured units into epi-illumination energy density (ED) for thesecond-energy form with units of J/cm² (i.e. Joules÷cm²):

Energy Density=Power×time÷area; or

Joules/cm² =Watts×Seconds÷cm² ; or

ED (J/cm²)=(mw÷1000)×(minutes×60)÷(0.000175 cm²); or

ED (J/cm²)=(342.86)×(mw)×(minutes)÷1 cm².

The above-described calibration procedure gives the followingepi-illumination energy densities (ED at the focal point of themicroscope objective lens) which result from use of the preferredepi-illumination neutral density (ND) filters:

    ______________________________________                                        ND     POWER (mw)      ED (Joules/cm.sup.2)                                   ______________________________________                                        0.0    0.40            137.14 × exposure mins                           0.1    0.32            109.71 × exposure mins                           0.3    0.20             68.57 × exposure mins                           0.6    0.10             34.29 × exposure mins                           ______________________________________                                    

Typical epi-illumination second-energy exposure times are 1 to 10minutes to give final gradations in the epi-illumination energy densityover a range of 34.29 to 1,371.40 J/cm² which provide gradations in thesecond-energy activation of the cell-attack agent to rupture cells asdifferent lysis rates in a microscopic area of a micro-sample on themicroscope stage.

The last step in microscope calibration is adjustment of the cameratarget-voltage when the cell micro-sample is first placed on themicroscope stage. This adjustment qualitatively positions the cameratarget-voltage (baseline target current) at approximately the midpointof the linear voltage range of the camera to give a reasonable picturefor the transilluminated first-energy image of the cell micro-sample.Quantitative measurement of this starting camera voltage is not requiredbecause subsequent changes in first-energy intensity at the cameratarget are measured as a percentage change in camera target current,which is independent of the starting target voltage when that targetvoltage is in the linear voltage range of the camera.

Cell Collection Procedure

Arterial, venous, or mixed capillary (finger stick) blood can be usedfor micro-lysis of red blood cells. The data in this patent report wereobtained from standard venipuncture of the antecubital vein in awakehumans and the ear vein of awake New Zealand white rabbits (2.5-4 kg) tocollect blood (typically 1-2 ml) into sterile tubes which containedcitrate to prevent clotting.

Blood is stored in a 0°-4° C. refrigerator before preparation of bloodspecimens and cell micro-samples. All cell and solution procedures useonly sterile disposable pipette tips, microcentrifuge tubes, bottles,stoppers, and labware to prevent cell contamination and infection.

Buffer Solution Preparation

Subsequent blood dilutions (described below) are done with a typicalbuffer solution consisting of 7.6 g/l NaCl, 0.26 g/l NaHPO₄. H₂ O, 1.28g/l Na₂ HPO₄, 4 g/l glucose, and 5 g/l bovine serum albumin. Buffersolution pH is adjusted to 7.4 (normal blood pH) by addition of 1N NaOH.The final osmolality of this buffer solution is 311 mOsm (near normalblood pH). Other buffer solutions can be used; however, glucose andalbumin are necessary ingredients to stabilize the cell preparation andto provide restricted-permeability molecules for control oftranscellular water movements which can otherwise affect the subsequentmicro-lysis process. Likewise, a final osmolality in the range of295-315 mOsm is critical since overall buffer solution osmolality alsoaffects the rapid phase of transcellular water movements across the cellmembrane.

The final step in buffer solution preparation is vacuum aspiration ofthe buffer solution through a sterile 0.2 micron Nalgene filter toremove any bacteria or other particulate contaminants. The filteredbuffer solution is allocated into sterile glass bottles (20 mlpreferred) with rubber stoppers for subsequent cell-specimenpreparation.

Cell-attack Solution Preparation

The type of energy-activated agent that is used for cell attack isspecific to the type of cell to be analyzed, and there is more than onetype of cell-attack agent which can be used for any one cell type. Forexample, phloxine B, eosin-isothiocyanate, pheophorbide, andprotoporphyrin have been used by others for photohemolysis of red bloodcells in a relatively large-volume cuvette. The preferred cell-attackagent for micro-lysis of red blood cell membranes is fluoresceinisothiocyanate (FITC) which is conjugated to a larger macromolecule(20,000 dalton dextran preferred). FITC-dextran is preferred because itdoes not enter the cell, and there is little blood absorption of thesecond-energy at the FITC-activation second-energy frequency of 480nanometers. Thus, the measured second-energy which is projected onto themicro-sample gives a precise amount of activated FITC as a cell-attackagent.

The FITC-dextran is mixed with a 0.9% NaCl solution to give anFITC-dextran concentration of 50 mg/ml. In the subsequent cellmicro-sample, the final FITC-dextran concentration in the micro-sampleshould be in the 2-8 mg/ml range to give the best energy-activatedcell-attack stimulus for micro-lysis of red blood cells.

Cell Specimen Preparation

Micro-lysis can be applied to different types of cells, and thecell-separation part of the cell preparation procedure will depend onthe cell type. The following describes the procedure for red bloodcells.

The refrigerated blood is inverted gently to mix the blood, and is thendiluted (1 to 1) with equal parts of the buffer solution. A small amount(80 to 100 μl ) of the diluted blood is drawn into a microhematocrittube which is spun in an IEC MB hematocrit centrifuge at the recommendedspeed to compact the red blood cells toward one end of themicrohematocrit tube. The length of the compacted red blood cells isthen measured to give the hematocrit (% cells) of the diluted blood.

The final cell-specimen is a combination of the diluted blood, thecell-attack agent FITC-dextran, a test chemical (if any), and the buffersolution to give a cell-specimen volume of 1 ml with a composition of0.5 to 6.0% red blood cells, 2.0 to 8.0 mg/ml FITC-dextran, 0 to 10mg/ml of the test chemical, and the remainder as buffer. As anillustration, a cell specimen with 4 mg/ml of FITC-dextran (which isprepared to beta 50 mg/ml FITC solution as described above), 3% redblood cells (which comes from a diluted blood solution with 25%hematocrit), and 3 mg/ml of a test chemical (which comes as a 20 mg/mlsolution) is obtained by mixing 0.08 ml (calculated as 4/50)of the FITCsolution, 0.12 ml (calculated as 3/25) of the diluted blood, 0.15 ml(calculated as 3/20) of the test chemical solution, and 0.65 ml(calculated as 1.0-0.08-0.12-0.15) of the buffer solution. Based on thisapproach, individual or combinations of soluble materials or chemicalscan be tested for an effect on cell response to micro-lysis by astandardized energy-activated cell-attack procedure.

Cell Micro-Sample Preparation

Any type of small volume (50 μl or less) chamber of transparentwater-impervious material, such as plastic or glass, can be used to givea cell micro-sample on the microscope stage. The crucial aspect is achamber and filling procedure that prevents micro-sample loss of water(which would change the cell micro-sample constituent concentrations toalter the micro-lysis procedure). The following describes a simple newmicro-sample preparation.

A hemacytometer (Neubauer preferred) and cover slip are sterilized inalcohol and air-dried. As a novel approach of the present invention,four Kerr absorbent points (dental cotton sticks for cavitypreparations) are placed in a 0.9% NaCl solution to saturate them. Afine forceps is then used to place two saturated Kerr points in eachside-well of the hemacytometer, with care to ensure that no Kerr pointtouches the inside edge of the hemacytometer trough (otherwise, thesolution in the Kerr points would dilute the micro-sample constituentconcentrations). The saturated Kerr points are a novel approach toprovide a "saturated" water vapor pressure to "replace" any potentialloss of micro-sample water to the atmosphere by evaporation through theentrance of the hemacytometer chamber.

A small drop-dispenser is used to pick up a solution of 2.5 g/l globulin(made in distilled water), and this solution is applied to coat thecoverslip rests of the hemacytometer. The coverslip is placed on thehemacytometer rests and is lightly tapped to seal the coverslip to theglobulin-coated hemacytometer rests. This is a novel approach of thepresent invention to reduce evaporation of water from the cellmicro-sample. In the customary approach by others, the coverslip isplaced on the hemacytometer rests without use of a sealant. Smallirregularities in the "rests" leave small openings between the coverslipand the "rests." The use of a globulin solution in the present inventionto seal these small openings is a novel approach to prevent evaporativewater loss from the cell micro-sample.

The cell micro-sample solution is gently mixed by inversion of thesolution vial. A sterile transfer pipette is used to aspirate the cellmicro-sample solution from the vial and to apply 22-25 μl of thesolution to the opening of the hemacytometer/coverslip chamber.Capillary action draws the cell micro-sample solution into thehemacytometer chamber (which has a preferred depth of 0.1 mm tostandardize the number of cell layers).

The hemacytometer is then placed in a sample carrier which is positionedon the microscope stage. At this point, the transilluminationfirst-energy pathway is opened (by removing the opaque filter from thefield limiter) and the cell micro-sample is brought into focus. Thetelevision camera target-voltage is adjusted to the midrange (describedabove in the microscope calibration procedure).

Micro-Lysis Cell-Attack Procedure

The television-microscope images in the following procedure are videorecorded for post-procedure analysis. (These images can also be analyzedon-line at the time of the procedure.)

A five-minute waiting period is used for cells to settle in themicro-sample hemacytometer chamber. Room temperature is recorded andhemacytometer temperature is kept at 23.5° to 24.5° C. to minimizeBrownian motion of the red blood cells in the cell micro-sample. Thecells are brought to focus during a first-energy transillumination togive a control reading of "background first-energy density" for the cellmicro-sample. Then, the transillumination pathway is closed by placingthe opaque filter on the field limiter.

The time-date video-insertion generator is reset to 0.00 time (inminutes) and is started at the instant that the second-energyepi-illumination pathway is opened (by removing an opaque filter fromthe epi-illumination pathway). The focus of the fluorescent image ischecked and readjusted if necessary. The second-energy epi-illuminationis left on for the necessary time (1 to 10 minutes preferred) with apreselected neutral density filter in the epi-illumination pathway togive a desired level of cell-attack activation which is measured as anepi-illumination second-energy density in Joules/cm² (as described abovein the microscope calibration procedure). At the end of the cell-attackactivation time, the epi-illumination pathway is closed (by placing theopaque filter in the epi-illumination pathway), and the first-energytransillumination pathway is opened (by removing the opaque filter onthe field limiter) to give cell micro-sample images. These cell imagesare recorded until lysis (rupture of cells) has reached a maximum (lessthan 5 intact cells remaining in the rectangular epi-illumination area)or 60 minutes has elapsed, whichever occurs first.

At this point, the hemacytometer cell micro-sample is repositionedmanually on the microscope stage to give another microscopic area of themicro-sample for application of another cell-attack activation in thesecond microscopic area. This procedure allows many measurements ofmicro-lysis at different cell-attack levels (epi-illumination energydensities) on each cell micro-sample. Hemacytometer grid etchings areused to identify the location of each microscopic cell-attack area whichprevents inadvertent overlap of cell-attack areas in multiplemicro-lysis procedures on the same cell micro-sample.

Micro Analysis Procedure

After the cell micro-sample (in the hemacytometer) has been placed onthe microscope stage, there is a five-minute period for cells to settle;in the micro-sample chamber. At this point, transillumination with thefirst-energy source gives a relatively dark speckled-image of themicro-sample in the television field of view. The speckled nature ofthis image is caused by the presence of red blood cells which absorb atthe first-energy frequency, as shown in FIG. 22.

This transillumination first-energy speckled-image area (which is 6 to10 times larger than the epi-illumination second-energy rectangulardiaphragm area) is digitized (Image Technology digitizing boardpreferred) to give a digital image with 512×512 pixels (1024×1024 wouldincrease analysis precision) with a digital value between 0 and 255 torepresent the energy intensity of each image pixel. The mean of ALLpixel values in this speckled first-energy image is calculated as amicro-sample "background first-energy intensity (BFEI)" with a valuebetween 0 and 255 (a typical value is 100 to 150).

As described above (section on Micro-Lysis Cell-Attack Procedure), theepi-illumination second-energy pathway is opened (by removal of theopaque filter) for a pre-selected time to activate a cell-attack agentwithin the epi-illumination rectangular microscopic area on the cellmicro-sample image. Then, the epi-illumination second-energy pathway isclosed. Transilluminated first-energy images of the micro-sample arethen digitized at 30-second intervals over the next 60 minutes (or untilthere are 5 or fewer intact cells within the rectangular cell-attackarea in the image).

After exposure to the epi-illumination second-energy rectangular area ofcell-attack activation, the Microscopic area of the cell micro-samplethat was exposed becomes brighter and loses its speckled appearance as afunction of time in the transilluminated first-energy image, as shown inFIG. 22. This occurs because cells in the microscopic area of thesecond-energy activated cell-attack lose part or all of their contentsand their membranes lose structural integrity as a micro-lysis responseto the second-energy activated cell-attack.

The values of the transilluminated first-energy image pixels in therectangular (approximately 60×50 pixels) microscopic cell-attack areaare averaged, and this pixel average is compared in successive digitizedimages for 60 minutes or until this pixel average rises to a plateauvalue which does not change by more than ±1% for 3 minutes (6 images).The transillumination first-energy image at 60 minutes, or the firsttransillumination micro-sample image with a plateau for the averagevalue of the first-energy image pixels in the rectangular microscopiccell-attack area, is defined as the maximal cell-response image. The 30highest pixel values (10% of total pixels) in the rectangularmicroscopic cell-attack area of the maximal cell-response image areaveraged to define a digital value which represents an image area withno intact cells (called the "total-lysis" first-energy intensity orTLFEI).

The average value for all pixels in the rectangular cell-attack area isdefined as a "Response-Area Energy Intensity (t)" for each digitizedtransilluminated first-energy micro-sample image. These Response-AreaEnergy Intensities (REI(t)) are converted to relative Response-AreaOptical Densities (Response OD(t)) by the following formula:

    Response OD(t)=100(TLFEI-REI(t))/(TLFEI-BFEI),

where TLFEI is the total-lysis first-energy intensity in the microscopiccell-attack area of the transilluminated micro-sample image, BFEI is thebackground first-energy intensity for the entire transilluminatedmicro-sample image, and REI(t) is the response first-energy intensity asa function of time in the microscopic cell-attack area of thetransilluminated micro-sample image. The quantity (TLFEI-BFEI)represents the range of the average light intensity for the entiretransilluminated first-energy micro-sample image. The zero-time averageenergy intensity of the rectangular microscopic area before cell-attack(REI(0)) can be larger or smaller than the average backgroundfirst-energy intensity (BFEI) for the entire transilluminatedfirst-energy micro-sample image. Thus, the zero-time Response-Area OD(0)can be larger or smaller than 100% of the optical density range for theentire transilluminated first-energy micro-sample image. The averageResponse-Area Energy Intensity in the maximal cell-response image (REIplateau) can be smaller than the total-lysis first-energy intensity(TLFEI) because some intact cells can remain in the rectangular area ofthe maximal response image. Thus, the minimum plateau Response-Area ODcan be larger than zero.

FIGS. 1 and 2 show the first-energy Response-Area optical densities as afunction of time from the beginning of a five-minute exposure of humanred blood cells to three levels of second-energy cell-attack activation(expressed as Joules per centimeter squared). A second-energycell-attack activation of 245 J/cm² gives a Response-Area OD whichdecreases from a zero-time value of 105% to a minimum plateau value of18% at 46 minutes after the beginning of the cell-attack activation(FIG. 1). A higher level of cell-attack activation of 401 J/cm² gives aResponse-Area OD which decreases from 102% to 6% at 31 minutes after thebeginning of cell-attack activation (FIG. 1), while an even greatercell-attack activation of 638 J/cm² gives a Response-Area OD whichdecreases from a zero-time value of 91% to a minimum plateau value of 3%at 37 minutes after the beginning of cell-attack activation (FIG. 2).These data show that changes in second-energy cell-attack activationalter the time-dependent first-energy Response-Area OD curve byaffecting the magnitude of the maximal OD change, the slope of theResponse-Area OD curve, and the time to the half-maximal change inResponse-Area OD.

The variation in zero-time first-energy Response-Area OD occurs beforeapplication of the second-energy cell-attack activation and addsstatistical variation to the time-dependent first-energy Response-AreaOD curve that is produced by the second-energy cell-attack activation.To remove the effect of this attack-unrelated variation in zero-timefirst-energy Response-Area OD, the following three-step normalizationprocedure is performed as illustrated in FIG. 3, wherein:

1) The Maximal Micro-Lysis Response (MR) is calculated as the zero-timeResponse-Area OD (ZROD) minus the Minimum Plateau Response-Area OD(PROD);

i.e. MR=ZROD-PROD.

2) The Percent Maximal Response (MR%) is calculated as 100 times theMaximal Micro-Lysis Response (MR) divided by the zero-time Response-AreaOD (ZROD);

i.e. MR%=(100)(MR)/(ZROD).

3) The % Response at a specific time t (% Response (t)) is defined as100 times the quantity, zero-time Response-Area. OD (ZROD) minus theResponse-Area OD at time t (ROD(t)), that resultant quantity divided bythe Maximal Micro-Lysis Response (MR);

i.e. % Response (t)=(100)(ZROD-ROD(t))/MR.

The Percent Maximal Response (MR%) is a measure of the percentage numberof cells which are eventually ruptured in the rectangular microscopicarea that was exposed to the second-energy cell-attack activation. The %Response (t) is a measure of the number of cells which have ruptured asof time t in the rectangular microscopic area that was exposed to thesecond-energy cell-attack activation, and is expressed as a percentageof the number of cells that are eventually ruptured.

As shown in FIG. 4, the three % Response curves are generated byapplication of the above formulas to the three Response-Area OpticalDensity curves in FIGS. 1 and 2. The above-described normalizationprocedure produces time-dependent % Response curves which change from 0%to 100% after the beginning of the cell-attack activation, irrespectiveof the variation in the zero-time Response-Area OD (ZROD in FIG. 3).These normalized data show that changes in cell-attack activation alterthe time-dependent % Response curves by changing the maximum slope andthe time to the half-maximal (50%) value of the curves.

Moreover, FIG. 5 repeats the % Response curve (open circles) of FIG. 4for the 245 Joules/cm² cell-attack activation to quantitate precisechanges in cell response to specific levels of second-energy cell-attackactivation by defining:

1) the response half-time (T_(1/2)) as the period from the beginning ofsecond-energy cell-attack activation to the time of the 50-percentresponse value, and

2) the largest slope of the % Response curve as the Slope at timet=T_(1/2).

T_(1/2) is a measure of average cell sensitivity to membrane rupture bythe second-energy cell-attack activation. The largest slope is a measureof the fastest rate of cell membrane rupture at any time after thebeginning of the cell-attack activation. This slope provides an index ofthe "population variation" in individual cell sensitivity to membranerupture by the cell-attack activation. For example, a population ofhomogeneous cells, each with exactly the same cell-sensitivity tomembrane rupture (i.e. same cell fragility), would give a "step" %Response curve with a zero-value until time equal to T_(1/2), and a 100%value after time equal to T_(1/2). In contrast, a very heterogeneouspopulation of cells, some with very low sensitivity and some with veryhigh sensitivity and some throughout the range from very low to veryhigh sensitivity to membrane rupture, would give a % Response curve witha shallow slope beginning shortly after the beginning of thesecond-energy cell-attack activation and reaching a 100% value at a timemuch after the T_(1/2) value.

The Slope of the % Response curve at time equal to T_(1/2) can beobtained in several ways. For example, the value of the first derivativeof a polynomial curve-fit to the % Response curve at time equal T_(1/2)would give the slope value of the % Response curve at time equal toT_(1/2). The following gives a simple method for calculation of theSlope of the % Response curve at time equal to T_(1/2) :

1) T70 is defined as the period (in minutes) from the beginning of thecell-attack activation to the time when the % Response equals 70%.

2) T30 is defined as the period (in minutes) from the beginning of thecell-attack activation to the time when the % Response equals 30%.

3) Slope at T_(1/2) is defined as 70% minus 30%, that quantity dividedby the quantity, T70 minus T30, with units of % Change in Response perMinute (abbreviated as %/MIN);

i.e. Slope at T_(1/2) (%/MIN)=(70-30)/(T70-T30).

This simple method is reasonably accurate because all % Response curvesrange from 0% to 100% in ordinate value, and all Response curves areapproximately linear in the 30% to 70% response range, such as the curvein FIG. 5 for example.

Micro-Lysis of Animal Cells

FIG. 6 shows the first-energy % Response curves (mean±SEM) on day 1 at 7hours (circles), day 2 (squares), day 3 (triangles), day 4 (diamonds),and day 5 (inverted triangles) after collection of red blood cells fromfour rabbits. The micro-sample composition was a 4% red blood cellconcentration and 2 mg/ml of the cell-attack agent FITC-dextran (150,000daltons), and the second-energy cell-attack activation was 210 J/cm² forall curves. The first-energy % Response curves are identical to eachother when micro-lysis is conducted on cell micro-samples from 7 hoursto 5 days after blood collection from rabbits. The % Response curve isshifted to the left of the curves in FIG. 6 when micro-lysis isconducted on cell micro-samples during the first 6 hours after bloodcollection from rabbits.

FIG. 7 shows first-energy % Response curves (mean±SEM) for the effect ofsecond-energy cell-attack activation of 105 Joules/cm² (open squares),126 Joules/cm² (filled squares), 159 Joules/cm² (open triangles), 168J/cm² (filled triangles), 210 J/cm² (open diamonds), and 210 J/cm²(filled diamonds) on rabbit (N=3) red blood cells with micro-samplecomposition of 4% hematocrit and 2 mg/ml of the cell-attack agentFITC-dextran (150,000 daltons). The second-energy cell-attackactivations for the open symbols were achieved by lower epi-illuminationpower densities for 10 minutes of second-energy exposure, while thecell-attack activations for the filled symbols were achieved by higherepi-illumination power densities for 8 minutes of second-energyexposure. The % Response curve for a lower epi-illumination powerdensity at a longer second-energy exposure time (open symbols) is thesame as that for a higher second-energy power density at a shortersecond-energy exposure time (filled symbols) when the two respective,power density multiplied by exposure time quantities, give the sameenergy densities to represent equivalent second-energy cell-attackactivations. This observation remains true for any second-energydensity, provided that the second-energy exposure time is at least 4minutes in duration. FIG. 7 also demonstrates that the Slope and T_(1/2)values of the % Response curves are different for different levels ofsecond-energy cell-attack activation. The % Response curves for rabbitred blood cells are also different for different micro-sampleconcentrations of the cell-attack agent FITC-dextran when theFITC-dextran concentration is below a critical value of 2.0 mg/ml.

FIG. 8 shows the first-energy % Response curves (mean±SEM) for 3%(circles), 4% (triangles), and 5% (squares) concentrations of rabbit(N=4) red blood cells in micro-samples that contained 2 mg/ml of thecell-attack agent FITC-dextran (150,000 daltons) and received acell-attack activation of 210 J/cm². These % Response curves for rabbitred blood cells are statistically the same for the differentmicro-sample cell concentrations in the 3 to 5% range, to show thatmoderate variation in cell concentrations will not change themicro-lysis measurement of cell sensitivity to membrane rupture bysecond-energy cell-attack activation.

FIG. 9 shows the first-energy % Response curves (mean±SEM) for rabbit(N=3) red blood cells that had been separated from whole blood (circles)and for red blood cells in whole blood that contained plasma and othercells such as platelets and white blood cells (triangles). Micro-samplecomposition was a 4% red blood cell concentration and 2 mg/ml of thecell-attack agent FITC-dextran (150,000 daltons). These data demonstratethat the first-energy % Response curves for rabbit red blood cells arenot affected by the presence of blood plasma or other blood cells (suchas white blood cells) in the micro-sample. Thus, the micro-lysisprocedure can be applied to whole blood samples, without the need forcareful separation of red blood cells.

FIG. 10 shows the first-energy % Response curves (mean±SEM) formicro-lysis of rabbit (N=4) red blood cells on day 5 (circles), on day 5after the red blood cells had been washed and then reconstituted withbuffer and the cell-attack agent FITC-dextran (diamonds), and on day 5after the red blood cells had been washed and then reconstituted withbuffer but not the cell-attack agent FITC-dextran (squares).Micro-sample composition was buffer, 2 mg/ml of FITC-dextran (150,000daltons), and a 4% red blood cell concentration, and the cell-attackactivation was 198 Joules/cm². Cells that were washed on day 5 andreconstituted without the cell-attack agent (FITC-dextran) did nothemolyze when exposed to the second-energy cell-attack activation.Washed cells reconstituted with the original cell-attack agent(FITC-dextran) had the same micro-lysis response as unwashed cells onday five, thus showing that the washed cells could still be affected bythe second-energy cell-attack activation but only in the presence of thecell attack agent (FITC-dextran). Thus, FIG. 10 shows that the presenceof a cell-attack agent is necessary for the micro-lysis process and thatother factors like heat from the second-energy source do not inducehemolysis in this method. FITC-dextran (150,000) is a large molecularweight neutral dextran that does not even traverse endothelial gapsbetween two cells under non-inflamed conditions. Since washed red bloodcells were unresponsive, the data in FIG. 10 demonstrate that themicro-lysis response is not the result of a non-specific second-energyinteraction with an intracellular or even intramembrane-bound chemical.The micro-lysis process with FITC-dextran as the cell-attack agent mustoccur by an interaction of the cell-attack agent and the outside of thecell membrane with subsequent rupture of the cell membrane.

FIG. 11 shows the first-energy % Response curves (mean±SEM) formicro-lysis of rabbit (N=4) red blood cells that had been prepared instandard buffer with glucose and albumin (squares), buffer with glucosebut not albumin (filled diamonds), buffer with albumin but not glucose(triangles), and buffer without albumin and glucose (circles). Allmicro-samples contained 2 mg/ml of the cell-attack agent FITC-dextran(150,000 daltons) and a 4% concentration of red blood cells, and wereexposed to a cell-attack activation of 180 J/cm² (right panel) and 90J/cm² (left panel). The % Response Curves for rabbit red blood cells arenot altered by removal of albumin from the cell micro-sample. Accordingto scientific literature, removal of albumin changes the shape(deformability) Of the red blood cell. Thus, these data show that themicro-lysis process measures cell characteristics other than those thatdepend solely on cell shape (i.e. deformability). The % Response curvesare shifted to the left to reflect greater cell sensitivity to astandardized cell-attack activation when glucose is removed from thecell micro-sample. Cells are very susceptible (an increased Slope anddecreased T_(1/2)) even to a very low cell-attack activation whenglucose is absent as illustrated in the left panel of FIG. 11. Accordingto scientific literature, glucose is necessary for cells to maintaintheir internal ATP (adenosine triphosphate) levels, and ATP is requiredfor cell metabolism to maintain the structure of the cell membrane.Thus, these data show that the micro-lysis process can detect areduction in cell membrane ability to maintain the structural integrityof the cell membrane (i.e. a change in cell fragility).

FIG. 12 shows the first-energy % Response curves (mean±SEM) formicro-lysis of rabbit (N=6) cell micro-samples that contained 2 mg/ml ofthe cell-attack agent FITC-dextran (150,000 daltons), standard buffer,and a 4% concentration of red blood cells which had been previouslyincubated for one hour with the test chemical diamide at a concentrationof 5.0 milliMolar (diamonds), 0.5 mM (squares), 0.05 mM (circles), or0.00 mM (filled triangles), with cell-attack activations of 180 J/cm²(right panel) and 90 J/cm² (left panel). The first-energy % Responsecurves for rabbit red blood cells are shifted to the left to reflectgreater cell sensitivity to a second-energy cell-attack activation whenthe micro-sample contains cells that have been previously incubated forone hour with the test chemical diamide. These diamide-incubated cellshave enhanced susceptibility (increased Slope and decreased T_(1/2)) toa very low cell-attack activation as illustrated in the left panel ofFIG. 12. According to scientific literature, diamide is a chemical whichoxidizes the sulfhydryl groups of proteins in the cell membrane toincrease the cross-linking of spectrin which disturbs the protein layersof the cell membrane to increase cell membrane fragility. Thus, thesedata show that the micro-lysis process can detect an increase in cellfragility which results from a disturbance to the protein layers of thecell membrane.

FIG. 13 shows the first-energy % Response curves (mean±SEM) formicro-lysis of rabbit (N=5) cell micro-samples that contained 2 mg/ml ofthe cell-attack agent FITC-dextran (150,000 daltons), standard buffer,and a 4% concentration of red blood cells which had been previouslyincubated for 20 minutes with the test anesthetic Chlorpromazine at amicroMolar concentration of 100 μM (circles), 30 μM (diamonds), 10 μM(squares), and 0 μM (filled triangles), with cell-attack activations of180 J/cm² (right panel) and 90 J/cm² (left panel). The first-energy %Response curves for rabbit red blood cells are shifted to the left toreflect greater cell sensitivity to a second-energy cell-attackactivation when the micro-sample contains cells that have beenpreviously incubated for 20 minutes with the test anestheticchlorpromazine. These chlorpromazine-incubated cells are also moresusceptible (decreased T_(1/2) but no change in Slope) to very lowlevels of cell-attack activation as illustrated in the left panel ofFIG. 13. According to scientific literature, chlorpromazine, in theconcentrations used here, primarily produces stomatocyte formationthrough an effect of chlorpromazine to alter the arrangement of thelipid bilayer in the cell membrane with less effect on the proteinlayers in the cell membrane. Thus, these data show that the micro-lysisprocess can detect an increase in cell membrane fragility which resultsprimarily from a disturbance to the lipid layers of the cell membrane.

FIG. 14 shows the first-energy % Response curves (mean±SEM) formicro-lysis of rabbit (N=6) cell micro-samples that contained 2 mg/ml ofthe cell-attack agent FITC-dextran (150,000 daltons), standard buffer,and a 4% concentration of red blood cells which had been previouslyincubated for one hour with a test chemical glutaraldehyde at aconcentration of 0.02% (circles with dashed line), 0.01% (diamonds),0.005% (triangles), 0.0025% (squares with dashed line), 0.00125%(circles), and 0.000% (filled triangles), with a cell-attack activationof 180 J/cm². The first-energy % Response curves for rabbit red bloodcells are shifted to the right with increased T_(1/2) and decreasedSlope to reflect less cell sensitivity to cell-attack activation whenthe micro-sample contains cells that have been previously incubated forone hour with a 0.01% or greater solution of the test chemicalglutaraldehyde. According to scientific literature, glutaraldehydedecreases membrane fragility (i.e. a less breakable cell) and increasesmembrane rigidity (i.e. a less deformable cell). Chlorpromazineincreases membrane fragility (i.e. more breakable) without any reportedchange in membrane rigidity (i.e. unchanged deformability). Diamideincreases membrane fragility (i.e. more breakable) and increasesmembrane rigidity (i.e. less deformable). Thus, comparison of the effectof these three test chemicals as shown in FIGS. 12, 13, and 14demonstrates that the micro-lysis process measures changes in cellmembrane fragility rather than simply cell membrane rigidity, and thatthe micro-lysis process can distinguish between a change in membranefragility due to altered lipid layers and a change due to alteredprotein layers.

A streptozoticin-injected laboratory rat is a widely-used animal modelof human insulin-dependent diabetes with elevated blood sugar(hyperglycemia) and glucose spill-over into the urine. Diet manipulationof a laboratory rat is a known animal model of humanhypercholesterolemia. FIG. 15 shows the Percent Maximal Response (MR%),the T_(1/2), and the Slope values of the first-energy % Response curvesfor micro-lysis of red blood cells from 5 normal control Sprague-Dawleylaboratory rats (C) at second-energy activations of 248±3.6 J/cm², 3streptozoticin-induced insulin-dependent diabetic Sprague-Dawley rats(D) at second-energy activations of 245±4.1 J/cm² and 6 diet-inducedhypercholesterolemic Sprague-Dawley rats (H) at second-energyactivations of 249±2.7 J/cm². All cell micro-samples had a compositionof 4 mg/ml of the cell-attack agent FITC-dextran (20,000 daltons),standard buffer, and a 3% concentration of red blood cells. Thefirst-energy % Response curves for red blood cells from normal rats,diabetic rats, and hypercholesterolemic rats have similar MaximalResponses to the micro-lysis process. Red blood cells from thehypercholesterolemic rats have normal T_(1/2) and Slope values. Cellsfrom the diabetic rats have significantly longer T_(1/2) and lower Slopevalues to indicate a substantial reduction in cell membrane fragilityfor the diabetic animals. These data demonstrate that the micro-lysisprocess can detect diseases (such as diabetes) which are thought toalter ion pumps (such as the calcium pumps) in the cell membrane.

Micro-Lysis of Human Cells

FIG. 16 shows the Percent Maximal Response (MR%) and the T_(1/2) andSlope values of the first-energy % Response curves for micro-lysis of0.5% to 3.0% concentrations of human (N=3) red blood cells inmicro-samples that contained 4 mg/ml of the cell-attack agentFITC-dextran (20,000 daltons). One subject received a cell-attackactivation of 549±0.3 J/cm², the second subject received 340±1.1J/cm²and the third subject received 198±1.3 J/cm² for all five cellconcentrations. Micro-lysis of red blood cells from humans gives thesame Percent Maximal Responses for micro-sample cell concentrations inthe 1.0% to 3.0% range, the same T_(1/2) values for cell concentrationsin the 0.5% to 2.0% range, and the same % Response curve Slopes for cellconcentrations in the 0.5% to 3% range as shown in FIG. 16. Micro-lysisof red blood cells from rabbits gives the same first-energy % Responsecurves for micro-sample cell concentrations in the 3% to 5 % range asillustrated in FIG. 8. These data indicate that the micro-lysis processis relatively insensitive to variations in cell concentrations withinspecific but different concentration ranges for animals and humans.

According to scientific literature, pentoxifylline, a therapeutic drugwhich increases peripheral blood flow in several human pathologies,increases the "flexibility" of cell membranes to permit easier passageof red blood cells through blood vessels; yet, pentoxifylline does notsimply alter cell membrane rigidity (cell deformability). FIG. 17 showsthe Percent Maximal Response (MR%) and the T_(1/2) and Slope values ofthe first-energy % Response curves for micro-lysis of human (N=5) cellmicro-samples that contained 4 mg/ml of the cell-attack agentFITC-dextran (20,000 daltons) and a 1% concentration of red blood cellswhich had been previously incubated at 4° C. FOR ONE HOUR with thetherapeutic drug pentoxifylline (PTFX) at concentrations of 10milliMolar (filled bars), 1 mM (striped bars), and 0 mM (clear bars).One subject received a second-energy cell-attack activation of 658±6.6J/cm² three subjects received 329±1.9 J/cm² and one subject received196±0.7 J/cm² for all three pentoxifylline concentrations. FIG. 18 showsthe Percent Maximal Response (MR%) and the T_(1/2) and Slope values ofthe first-energy % Response curves for micro-lysis of human (N=5) cellmicro-samples that contained 4 mg/ml of the cell-attack agentFITC-dextran (20,000 daltons) and a 1% concentration of red blood cellswhich had been previously incubated at 4° C. FOR TWENTY-FOUR HOURS withthe therapeutic drug pentoxifylline (PTFX) at concentrations of 10milliMolar (filled bars), 1 mM (striped bars), and 0 mM (clear bars).One of these subjects received a second-energy cell-attack activation of641±3.3 J/cm² three subjects received 330±1.7 J/cm² and one subjectreceived 205±0.7 J/cm² for all three pentoxifylline concentrations.Incubation of human red blood cells with pentoxifylline for one houralters the micro-lysis response of those cells. As the pentoxifyllineconcentration is increased, there is a progressive (dose-dependent)decrease in the Percent Maximal Response, an increase in T_(1/2), and adecrease in the Slope of the micro-lysis % Response curves asillustrated in FIG. 17. With 24-hour pentoxifylline incubation of redblood cells as shown in FIG. 18, the decrease in the Percent MaximalResponse is lost, and the decrease in Slope is partially lost, but thedose-dependent increase in T_(1/2) is still present. These datademonstrate that the micro-lysis process is sensitive to drug-inducedalterations in human cell membranes as a function of time after drugtreatmentor and that micro-lysis can distinguish between drug-treatedand non-treated human cells.

FIG. 19 shows the Percent Maximal Response (MR%) and the T_(1/2) andSlope values of the first-energy % Response curves at second-energycell-attack activations of A=243±2.9 J/cm² (clear bars), B=400±3.6 J/cm²(striped bars), and C=642±4.0 J/cm² (filled bars) for micro-lysis of redblood cells from four female and three male African-Americans who had noclinical indicators of sickle-cell disease. The cell micro-samplescontained 4 mg/ml of the cell-attack agent FITC-dextran (20,000daltons), a 3% concentration of red blood cells, and buffer. FIG. 20shows the Percent Maximal Response (MR%) and the T_(1/2) and Slopevalues of the first-energy % Response curves at second-energycell-attack activations of A=247±1.1 J/cm² (clear bars), B=395±5.5 J/cm²(striped bars), and C=643±4.0 J/cm² (filled bars) for micro-lysis ofred,blood cells from 2 female and 3 male African-Americans with clinicaldiagnosis of sickle-cell disease but not hemoglobin-F abnormality. Thecell micro-samples contained 4 mg/ml of the cell-attack agentFITC-dextran (20,000 daltons), a 3% concentration of red blood cells,and buffer. Normal red blood cells from humans without sickle-celldisease as illustrated in the left panel of FIG. 19 show an eventual 88to 95% cell rupture (Percent Maximal Response, MR%) after exposure tocell-attack activations across a broad range from 240 J/cm² to 660J/cm². These normal human cells have micro-lysis % Response curves witha progressively decreasing T_(1/2) and a progressively increasing Slopeas the second-energy cell-attack activation is increased as shown inFIG. 19. In contrast, red blood cells from humans with sickle-celldisease have a substantially reduced Percent Maximal Response (with apeak at an intermediate cell-attack activation level), a significantlyelevated T_(1/2) which is not a function of the cell-attack activationlevel, and a substantially reduced Slope as is evident in a comparisonof FIG. 20 to FIG. 19. These data demonstrate that the micro-lysisprocess is sensitive to pathologic alterations in human cell membranes,and that micro-lysis provides multiple parameters for detection of humandiseases that alter cells.

FIG. 21 shows the Percent Maximal Response (MR%) and the T_(1/2) andSlope values of the first-energy % Response curves at second-energycell-attack activations of A=253±2.8 J/cm² (clear bars), B=399±4.6 J/cm²(striped bars), and C=661±2.1 J/cm² (filled bars) for micro-lysis of redblood cells from 1 female and 1 male African-American with clinicaldiagnosis of sickle-cell disease and hemoglobin-F abnormality. The cellmicro-samples contained 4 mg/ml of the cell-attack agent FITC-dextran(20,000 daltons), a 3% concentration of red blood cells, and buffer. Redblood cells from humans with a combination of sickle-cell disease and ahemoglobin-F abnormality have a normal Percent Maximal Response, anabnormally high T_(1/2), and a normal Slope at lower cell-attackactivations but a reduced Slope at the higher cell-attack activation asis evident in a comparison of FIG. 21 to FIG. 19. These data show thatthe micro-lysis process is sensitive to combinations of pathologicalterations in human cell membranes. Also these data demonstrate thatmultiple parameters of the micro-lysis process can differentiate betweennormal human cells as shown in FIG. 19 and single-disease altered cellsas shown in FIG. 20, and can distinguish both of those situations frommultiple abnormalities such as those shown in FIG. 21.

The foregoing detailed description is given primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom, for modifications will become obvious to those skilled in theart based upon more recent disclosures and may be made without departingfrom the spirit of the invention and scope of the appended claims.

I claim:
 1. A micro lysis-analysis process for measuring the strength ofa cell membrane and detecting abnormalities thereof by applying precisequantities of a first focused photo energy of a selected frequency andenergy density to a precise microscopic area of a cell sample containingtest cells for measuring as a function of time the precise degree ofcell disruption, applying a second focused photo energy of a secondselected frequency and energy density to said cell sample therebyphoto-activating a photo-activated cell-attack agent for attacking cellmembranes in said microscopic area of photo-stimulated cell-attackproviding a quantitative, time-related, photo-dose dependent measure ofcell fragility, comprising the steps of:suspending test cells whosemembrane strength is to be measured in a solution forming a cellcontaining solution; adding an inactive photo-activated cell-attackagent to said cell containing solution, said inactive cell-attack agentcomprising molecules absorbing photo energy in a known range ofexcitation frequencies producing a photosensitive chemical solution;preparing a micro-sample of said photosensitive chemical solutioncontaining said test cells; identifying a localized microscopic areawithin said micro-sample of said photosensitive solution; applying aspecific amount of the first focused photo energy in a cell absorptionrange to said micro-sample of said photosensitive chemical solutionexposing said cells in said micro-sample to said first focused photoenergy for a selected period of time for determining photo energyabsorption by intact test cells in said localized microscopic area ofsaid micro-sample at any time period; determining the photo energyabsorption and number of intact test cells in said localized microscopicarea by measuring the amount of said first focused photo energy passingthrough said test cells in said micro-sample of said photosensitivechemical solution in said localized microscopic area; applying aspecific amount of the second focused photo energy for a selected timeat a second selected wavelength, said second focused photo energy beingcapable of exciting said inactive cell-attack agent, and said secondfocused photo energy photo-activating said photosensitive chemicalsolution forming a cell-reacting medium in said localized microscopicarea containing said test cells; reacting said cell-reacting mediumcontaining said test cells to disrupt cell membranes of said test cellspermitting loss of said cell contents and changing the absorption ofsaid first focused photo energy by said test cells in said localizedmicroscopic area: analyzing the strength of said cell membranequantitatively by comparing the amount of said first focused photoenergy passing through said test cells in said micro-sample aftermicro-sample exposure to said second focused photo energy, with theamount of said first focused photo energy passing through said testcells in said micro-sample before micro-sample exposure to said secondfocused photo energy for determining photo energy absorption byremaining intact test cells in said localized microscopic area atselected time intervals; and coordinating and comparing the analyzedcell membrane strength to values for normal cells to measure changes incell fragility.
 2. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 1, wherein saidanalyzing step comprises comparing a starting number of cells with themaximum number of test cells disrupted, the time to reach disruption of1/2 of the maximum number of test cells to be disrupted, and a maximumrate of cell disruption during said selected time intervals.
 3. Themicro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 1, wherein said analyzing step comprisescomparing the starting number of test cells with the maximum number oftest cells disrupted, the time to reach disruption of 1/2 of the maximumnumber of test cells to be disrupted, and a maximum rate of celldisruption.
 4. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 1, including the step ofselecting said test cells from the group consisting of red cells, whitecells, platelets, and mixture thereof.
 5. The micro lysis-analysisprocess for measuring the strength of a cell membrane as claimed inclaim 1, including the step of suspending test cells whose membranestrength are to be measured in a physiologic salt solution of selectedosmolality forming a cell containing solution.
 6. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, including the step of suspending test cells whosemembrane strength is to be measured in a physiologic solution ofselected osmolality ranging from about 295 to about 315 milliosmolesforming a cell containing solution.
 7. The micro lysis-analysis processfor measuring the strength of a cell membrane as claimed in claim 1,including the step of applying said first focused photo energy in timeintervals ranging from about 2 minutes to about 60 minutes.
 8. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, including the step of applying said second focusedphoto energy in time intervals ranging from about 2 minutes to about 10minutes.
 9. The micro lysis-analysis process for measuring the strengthof a cell membrane as claimed in claim 1, including the step of applyingand measuring the amount of said first focused photo energy passingthrough said micro-sample at 30-second time intervals over a 60-minuteperiod or until there are 5 or fewer intact test cells within saidlocalized microscopic area.
 10. The micro lysis-analysis process formeasuring the strength of a cell membrane as claimed in claim 1,including the step of preparing a micro-sample of about 25 microliters(μl) of said photosensitive chemical solution.
 11. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, including the step of applying a second focusedphoto energy to photo-activate said inactive cell-attack agent fromabout 2 minutes to about 10 minutes depending on the particular inactivecell-attack agent selected.
 12. The micro lysis-analysis process formeasuring the strength of a cell membrane as claimed in claim 1,comprising the step of adding a test substance to said cell containingsolution providing test substance induced alterations in cell membranesfor determining the effect of said test substance on cell response tomicro lysis by applying a standardized photo-activated cell-attack agentand distinguishing between test substance treated and nontreated cellsas a function of time after test substance treatment, said testsubstance being selected from the group consisting of glutaraldehyde,albumin, chlorpromazine, diamide, glucose, albumin, and pentoxyifylline.13. The micro lysis-analysis process for measuring the strength of acell membrane as claimed in claim 1, further including the step ofadding diamide to said cell containing solution for oxidizing sulfhyrylgroups of proteins in the cell membrane to increase membrane fragilityand for increasing cross-linking of spectrin to increase membranerigidity and disrupt cell membrane integrity inducing alterations incell membranes for determining the effect of said diamide on cellresponse to micro-photo lysis by applying a standardized photo-activatedcell-attack agent and distinguishing between diamide-treated andnontreated cells as a function of time after diamide treatment.
 14. Themicro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 1, further including the step of addingchlorpromazine to said cell containing solution producing stomatocytesand altering a lipid bilayer of said cell membrane to disrupt cellmembrane integrity by increasing membrane fragility without any changein rigidity of said cell membrane inducing alterations in cell membranesfor determining the effect of said chlorpromazine on cell response tomicro lysis by applying a standardized photo-activated cell-attack agentand distinguishing between chlorpromazine treated and nontreated cellsas a function of time after chlorpromazine treatment.
 15. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, further including the step of adding glutaraldehydeto said cell containing solution decreasing membrane fragility andincreasing membrane rigidity of said cell membrane inducing alterationsin cell membranes for determining the effect of said glutaraldehyde oncell response to micro lysis by applying a standardized photo-activatedcell-attack agent and distinguishing between glutaraldehyde treated andnontreated cells as a function of time after glutaraldehyde treatment.16. The micro lysis-analysis process for measuring the strength of acell membrane as claimed in claim 1, further including the step ofcomparing the effect of adding at least one test substance to said cellcontaining solution for measuring the effect of said test substance onchanges in said cell membrane fragility and distinguishing between achange in said membrane fragility due to altered lipid layers and achange in said membrane fragility due to altered protein layers of saidcell membrane.
 17. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 1, further comprisingthe step of selecting human red blood cells as said test cells andcomparing said selected red blood cells with control cells for detectingdiabetes by changes in the cell-response to micro lysis for changes inmembrane integrity caused by diabetes.
 18. The micro lysis-analysisprocess for measuring the strength of a cell membrane as claimed inclaim 1, further comprising the step of determining the level of ATP(adenosine triphosphate) in a cell sample by treating the cell samplewith known concentrations of said ATP inducing alterations in cellmembranes of the test cells for determining the effect of said additionof said ATP on cell response to micro lysis by applying a standardizedphoto-activated cell-attack agent to the test cells and distinguishingbetween the integrity of the ATP affected cells and untreated cellsbefore and after the addition of ATP as a function of time aftertreatment and comparing the membrane integrity of cells in the untreatedtest cells with the membrane integrity of test cells in the ATP-treatedcell containing solution.
 19. The micro lysis-analysis process formeasuring the strength of a cell membrane as claimed in claim 1, furthercomprising the step of selecting human red blood cells as said testcells and comparing said selected red blood cells with control humanblood cells for detecting sickle-cell anemia by changes in thecell-response to micro lysis for changes in cell membrane integritycaused by said sickle-cell anemia.
 20. The micro lysis-analysis processfor measuring the strength of a cell membrane as claimed in claim 1,further including the step of selecting a specific photo-activatedcell-attack agent for a specific type of test cell.
 21. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, including the step of standardizing the osmolalityof said test cells and photo-activated cell-attack agent in astandardized buffer solution.
 22. The micro lysis-analysis process formeasuring the strength of a cell membrane as claimed in claim 1,including the step of varying the concentration of said test cellssuspended in said cell containing solution.
 23. The micro lysis-analysisprocess for measuring the strength of a cell membrane as claimed inclaim 1, which comprises the steps of testing for a cell membranealtering disease including diabetes, hypercholesterolemia, andsickle-cell anemia.
 24. The micro lysis-analysis process for measuringthe strength of a cell membrane as claimed in claim 1, further includingthe step of adding an intracellular membrane-bound specific test drug tosaid cell containing solution for measuring alterations in cell membranefragility due to changes in membrane characteristics that are created bya photo-activated cell-attack agent that passes through the cellmembrane to attach to internal cell structures where said cell attackagent becomes activated only inside of the test cells.
 25. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, further including the step of adding anextracellular membrane-bound specific test drug to said cell containingsolution for measuring alterations in cell membrane fragility due tochanges in membrane characteristics that are created by aphoto-activated cell-attack agent that remains and becomes activatedonly outside the test cells.
 26. The micro lysis-analysis process formeasuring the strength of a cell membrane as claimed in claim 1, furtherincluding the step of preparing a micro-sample of about 25 microliters(μl) of said photosensitive chemical solution within a covered wellmeans and placing at least one water saturated wick within said wellmeans in close proximity to said micro-sample for preventing dehydrationof said micro-sample of said photosensitive chemical solution.
 27. Themicro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 1, including the step of producing acell-response curve having at least four different quantitativemeasurements of cell membrane integrity for said selected time intervalsof cell response to a photo-activated cell-attack agent.
 28. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 1, further comprising the step of determining the levelof glucose in the cell containing solution by treating the cellcontaining solution with known amounts of glucose and comparing themembrane integrity of test cells in the untreated cell containingsolution with the membrane integrity of test cells in the treated cellcontaining solution.
 29. A micro lysis-analysis process for measuringthe strength of a cell membrane as claimed in claim 1, wherein saidphoto energy is in a range of about 200 to about 700 nanometers.
 30. Themicro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 1, further including the step of selectingsaid photo-activated cell-attack agent specific to the type of cell tobe analyzed.
 31. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 30, further includingthe step of selecting said photo-activated cell-attack agents from thegroup consisting of phloxine B, eosin-isothiocyanate, pheophorbide,protoporphyrin, fluorescein isothiocyanate, fluorescein isothiocyanatedextran, and combinations thereof.
 32. The micro lysis-analysis processfor measuring the strength of a cell membrane as claimed in claim 1,wherein said cell-attack agent is a photo-activated cell-attackfluorochrome.
 33. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 32, further includingthe step of selecting said photo-activated cell-attack fluorochrome fromthe group consisting of phloxine B, eosin-isothiocyanate, fluoresceinisothiocyanate, fluorescein isothiocyanate dextran, and combinationsthereof.
 34. The micro lysis-analysis process for measuring the strengthof a cell membrane as claimed in claim 1, further comprising the step ofselecting human red blood cells as said test cells and comparing saidselected red blood cells with control human blood cells for detectingpathologic alterations in human cell membranes by changes in thecell-response to micro lysis for changes in the cell membrane integritycaused by said pathologic alterations.
 35. The micro lysis-analysisprocess for measuring the strength of a cell membrane as claimed inclaim 34, further comprising the step of selecting human red blood cellsas said test cells and comparing said selected red blood cells withcontrol human blood cells for differentiating between normal human cellsand single-disease altered cells and for distinguishing said normalhuman cells and said single-disease altered cells from multiple-diseasealtered cells.
 36. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 1, including the step ofapplying said second focused photo energy to produce a pattern ofchanges for untreated normal cells with normal membranes and normalmetabolism and comparing the strength of the membrane of said untreatednormal cells with the strength of the membrane of cells which have beentreated by exposure to a known chemical.
 37. The micro lysis-analysisprocess for measuring the strength of a cell membrane as claimed inclaim 36, which comprises the steps of adding a chemical which alters acell membrane including pentoxifylline, diamide, glutaraldehyde, andchlorpromazine.
 38. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 1, further including thestep of adding a test drug to said cell containing solution providingdrug-induced alterations in cell membranes for determining the effect ofsaid test drug on cells response to micro lysis by applying astandardized photo-activated cell-attack agent and distinguishingbetween drug-treated and nontreated cells as a function of time afterdrug treatment.
 39. The micro lysis-analysis process for measuring thestrength of a cell membrane as claimed in claim 38, wherein said testdrug is an intracellular-bound specific test drug.
 40. The microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 38, further comprising the steps of detectingdrug-induced alterations in human cell membranes of a cell samplecontaining an unknown concentration of a drug by adding knownconcentrations of said drug to a cell containing solution inducingalterations in said cell membrane for determining the effect of saidaddition of known concentration of said drug on cell response to microlysis by applying a standardized photo-activated cell-attack agentbefore and after the addition of the known concentration of drug.
 41. Amicro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 1, including the step of obtaining multiplecell fragility measurements in a single micro-sample by independentlyreacting said cell attack agent in multiple separated microscopic areasof a single micro-sample and leaving other microscopic areas in themicro-sample as baseline for unactivated cell-attack sample references.42. A micro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 41, wherein said cell containing solutionfurther contains other cells of a different type than said test cells.43. A micro lysis-analysis process for measuring the strength of a cellmembrane as claimed in claim 42, wherein said cell containing solutioncontains different types of cells from blood samples.
 44. A microlysis-analysis process for measuring the strength of a cell membrane asclaimed in claim 43, wherein said different type of cells includes redblood cells, white blood cells, and platelets.